Carol L. Stone

Purification and characterization of a human liver cocaine carboxylesterase that catalyzes the production of benzoylecgonine and the formation of cocaethylene from alcohol and cocaine.

The psychomotor stimulant cocaine is inactivated primarily by hydrolysis to benzoylecgonine, the major urinary metabolite of the drug. A non-specific carboxylesterase was purified from human liver that catalyzes the hydrolysis of the methyl ester group of cocaine to form benzoylecgonine. In the presence of ethanol, the enzyme also catalyzes the transesterification of cocaine producing the pharmacologically active metabolite cocaethylene (benzoylecgonine ethyl ester). The carboxylesterase obeys simple Michaelis-Menten kinetics with Km values of 116 microM for cocaine and 43 mM for ethanol. The enzymatic activity suggests that it may play an important role in regulating the detoxication of cocaine and in the formation of the active metabolite cocaethylene. Additionally, the enzyme catalyzes the formation of ethyloleate from oleic acid and ethanol. The carboxylesterase was purified from autopsy liver by gel filtration, chromatofocusing, ion-exchange, and hydrophobic interaction chromatography to purity by SDS-PAGE and agarose gel isoelectric focusing. The subunit molecular weight was determined to be 59,000 and the native molecular weight was estimated to be 170,000 from a calibrated gel filtration column, suggesting that the active enzyme is a trimer. The isoelectric point was approximately 5.8. Digestion of carbohydrate residues on the protein with an acetylglucosaminidase plus binding to several lectins indicates that the enzyme is glycosylated. The esterase was cleaved with two proteases, and the amino acid sequences from fourteen peptides were used to search GenBank. Two identical matches were found corresponding to carboxylesterase cDNAs from human liver and lung.

cDNA Sequence and Catalytic Properties of a Chick Embryo Alcohol Dehydrogenase That Oxidizes Retinol and 3β,5α-Hydroxysteroids

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of ∼2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver λgt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of ∼40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min−1 for ethanol at pH 7.4 and 25°C. ADH-F was stereospecific for the 3β,5α- versus 3β,5β-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 μM for all-trans-retinol and 31 μM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

X-ray structure of human beta3beta3 alcohol dehydrogenase. The contribution of ionic interactions to coenzyme binding.

The three-dimensional structure of the human β3β3 dimeric alcohol dehydrogenase (β3) was determined to 2.4-Å resolution. β3 was crystallized as a ternary complex with the coenzyme NAD+ and the competitive inhibitor 4-iodopyrazole. β3 is a polymorphic variant at ADH2 that differs from β1 by a single amino acid substitution of Arg-369 → Cys. The available x-ray structures of mammalian alcohol dehydrogenases show that the side chain of Arg-369 forms an ion pair with the NAD(H) pyrophosphate to stabilize the E·NAD(H) complex. The Cys-369 side chain of β3 cannot form this interaction. The three-dimensional structures of β3 and β1 are virtually identical, with the exception that Cys-369 and two water molecules in β3 occupy the position of Arg-369 in β1. The two waters occupy the same positions as two guanidino nitrogens of Arg-369. Hence, the number of hydrogen bonding interactions between the enzyme and NAD(H) are the same for both isoenzymes. However, β3 differs from β1 by the loss of the electrostatic interaction between the NAD(H) pyrophosphate and the Arg-369 side chain. The equilibrium dissociation constants of β3 for NAD+ and NADH are 350-fold and 4000-fold higher, respectively, than those for β1. These changes correspond to binding free energy differences of 3.5 kcal/mol for NAD+ and 4.9 kcal/mol for NADH. Thus, the Arg-369 → Cys substitution of β3 isoenzyme destabilizes the interaction between coenzyme and β3 alcohol dehydrogenase.

Role of Alcohol Dehydrogenases in Steroid and Retinoid Metabolism

Retinoid and steroid hormones play an important role in the regulation of differentiation and maintenance of a wide range of animal tissues. These tissues include reproductive organs, liver, kidney, heart, brain, and skin of species from fish to humans. Several isozymes of cytosolic NAD+-dependent 40 kDa subunit molecular weight alcohol dehydrogenases catalyze oxidation and reduction of retinoid and steroid substrates in vitro. The isozymes are grouped into classes based on the similarities in amino acid sequence and their substrate specificities. Currently, a total of six classes of mammalian ADHs are known (Jornvall and Hoog, 1995). Each class has a characteristic tissue-specific and developmental pattern of expression (Edenberg and Bosron, 1996). Class I ADHs are basic isozymes with a wide range of Km for ethanol (0.05–36 mM). In humans, class I is comprised of multiple molecular forms, β1β1, β2β2, β3β3, γ1γ1, γ2γ2, α α, and their heterodimers. During development, ᾲᾳ is the first ADH isozyme detectable in fetal liver. β-ADH appears by mid-gestation, and γ-ADH is first detected about six month after birth. Human class II π-ADH has a relatively high KM for ethanol (34 mM) and is found in fetal and adult liver. The ubiquitously expressed class III ADH, also known as glutathione-dependent formaldehyde dehydrogenase, is not saturable with ethanol and is not active with either steroid or retinoid alcohols. Human class IV σ-ADH exhibits high KM for ethanol (28 mM) and is present in the adult stomach, esophagus and epithelium. In mice embryos, class IV ADH is detected on day 7.5 of development in the craniofacial region as well as trunk and forelimb bud mesenchyme (Ang, H.L. et al., 1996). Little is known about the catalytic properties of human class V and deermouse class VI ADH isozymes.

KINETICS OF A GLYCINE FOR ARG-47 HUMAN ALCOHOL DEHYDROGENASE MUTANT CAN BE EXPLAINED BY LYS-228 RECRUITMENT INTO THE PYROPHOSPHATE BINDING SITE

Ethanol and other alcohols are metabolized in liver by alcohol dehydrogenase isoenzymes. At least five electrophoretically distinct isoenzymes are found in human liver that can be divided into three classes (Burnell & Bosron, 1989). Class I comprises the a, β, and γ subunits, while class II contains the π subunit and class III contains the χ subunit. In addition, heterogeneity exists at the β and γ loci, producing the β1, β2, and β3 subunits, and the γ1 and γ2 subunits. Although the class II and III subunits associate only with themselves to form active homodimers, the class I isoenzymes associate to form both homodimers and heterodimers. Thus, the liver can contain as many as 17 active alcohol dehydrogenase isoenzyme forms.

Cloning and expression of a human stomach alcohol dehydrogenase isozyme

In humans, there is a family of NAD+- and zinc-dependent alcohol dehydrogenases (E.C. 1.1.1.1) that exhibit broad substrate specificity toward aliphatic alcohols (Vallee and Bazzone, 1983, Smith, 1986, and Bosron et al., 1993). The various isozyme subunits are encoded by at least 6 different gene loci (ADH1 through ADH6). Most recently, a new isozyme called σ-ADH or μ-ADH has been isolated from human stomach tissue that has a high K m (about 30 mM) and relatively high catalytic efficiency for ethanol (k c /K m ∼ 52 min-1mM-1)(Wang et al., 1990, Stone et al, 1993, Moreno and Pares, 1991). One or more isozymes with similar electrophoretic mobility are found in the esophagus (Yin et al., 1990).